Methods for developing endothelial cells from pluripotent cells and endothelial cells derived

Abstract

Disclosed herein is a method for developing human endothelial cells (ECs) from human embryonic stem cells (ESCs). The method is based on inhibition of TGF signaling following mesoderm induction and during vascular differentiation of hESC-derived cells. Also disclosed herein is a substantially pure and stable population of ECs that maintains a high degree of proliferation and phenotypic homogeneity for extended culture periods. Related pharmaceutical compositions and therapeutic methods are also disclosed. A reporter hESC line useful for tracking the development of ECs is also provided.

1 . A method for developing human endothelial cells (ECs) from human pluripotent cells in culture, comprising:
 forming embryoid bodies (EBs) from said pluripotent cells;   culturing the EBs under conditions sufficient for mesoderm induction;   culturing the cells under conditions that promote vascular differentiation and generation of ECs in the cell population; and   further culturing the cells in the presence of a TGFβ signaling inhibitor to expand ECs in the cell population.   
     
     
         2 . The method of  claim 1 , wherein said pluripotent cells are embryonic stem cells (ESCs). 
     
     
         3 . The method of  claim 1 , wherein said pluripotent cells are induced pluripotent cells. 
     
     
         4 . The method of  claim 2 , wherein EBs are formed by culturing the human ESCs in media devoid of FGF-2 and supplemented with a BMP. 
     
     
         5 . The method of  claim 1 , wherein the step of culturing the EBs under conditions sufficient for mesoderm induction comprises culturing the EBs in media supplemented with an activin and a BMP, and further culturing in media additionally supplemented with FGF-2. 
     
     
         6 . The method of  claim 5 , wherein the activin is activin A, the BMP is BMP4. 
     
     
         7 . The method of  claim 6 , wherein the EBs are cultured for 2 days in the presence of activin A and BMP4, and further cultured for 2-3 days in the presence of activin A, BMP4 and FGF-2. 
     
     
         8 . The method of  claim 1 , wherein the step of culturing the cells under conditions that promote vascular differentiation and generation of ECs comprises culturing the cells on an adherent substrate in media supplemented with VEGF-A, a BMP and FGF-2. 
     
     
         9 . The method of  claim 8 , wherein the cells are cultured for 3-4 days. 
     
     
         10 . The method of  claim 1 , wherein said TGFβ signaling inhibitor is an inhibitor specific for the type I TGFβ receptors. 
     
     
         11 . The method of  claim 10 , wherein said inhibitor inhibits signaling mediated by ALK4, ALK5, and ALK7. 
     
     
         12 . The method of  claim 10 , wherein said inhibitor is an inhibitor of at least ALK5. 
     
     
         13 . The method of  claim 10 , wherein said inhibitor is a polypeptide comprising a soluble form of a type I TGFβ receptor, an antibody directed to a type I TGFβ receptor, or a small molecule compound. 
     
     
         14 . The method of  claim 10 , wherein said inhibitor is a small molecule compound selected from SB-431542, A 83-01, D 4476, LY 364947, SB 525334, SD 208, and SJN 2511. 
     
     
         15 . The method of  claim 14 , wherein said inhibitor is SB-431542. 
     
     
         16 . The method of  claim 1 , wherein ECs are expanded in the presence of said TGFβ signaling inhibitor, VEGF-A and FGF-2. 
     
     
         17 . The method of  claim 1 , further comprising isolating expanded ECs from the cell population. 
     
     
         18 . The method of  claim 17 , further comprising culturing the isolated ECs in media supplemented with said TGFβ signaling inhibitor, VEGF-A and FGF-2. 
     
     
         19 . A substantially pure population of hESC-derived ECs, wherein said ECs are characterized by expression of surface markers, VE-cadherin, CD31 and VEGFR2. 
     
     
         20 . A composition comprising the substantially pure population of ECs of  claim 19  and at least one pharmaceutically acceptable carrier or diluents. 
     
     
         21 . A method for repairing injured tissue in a human subject, comprising administering to the subject the composition of  claim 20  to promote vascularization in said tissue. 
     
     
         22 . A method for treating a tumor in a human subject, comprising administering to the subject the composition of  claim 20 , wherein said ECs are engineered to deliver an anti-tumor agent, and upon administration, said ECs form vessels into said tumor. 
     
     
         23 . A human ESC reporter cell line, said cell line stably transfected with a nucleic acid molecule coding for a fluorescent protein, operably linked to the promoter region of the VE-cadherin gene. 
     
     
         24 . The reporter cell line of  claim 23 , wherein said fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), blue fluorescent protein (BFP), mOrange fluorescent protein, mCherry fluorescent protein, and yellow fluorescent protein (YFP).